Abstract
Accurate detection of gene sequences in single cells is the ultimate challenge to polymerase chain reaction (PCR) sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of linear-after-the-exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at different concentrations, but with novel design criteria to ensure high efficiency and specificity. Compared with conventional PCR, LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATEPCR signals provides a means for improving the accuracy of single cell genetic diagnosis.
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References
Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.
Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real Time Quantitative PCR. Genome Res. 6, 86–94.
Kostrikis, L. G., Tyagi, S., Mhlanga, M. M., Ho, D. D., and Kramer, F. R. (1998) Spectral genotyping of human alleles. Science 27, 1228, 1229.
Gyllensten, U. B. and Erlich, H. A. (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc. Natl. Acad. Sci. USA 85, 7652–7656.
Sanchez, J. A., Pierce, K. E., Rice, J. E., and Wangh, L. J. (2004) Linear-After-The-Exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proc. Natl. Acad. Sci. USA 101, 1933–1938.
Pierce, K. E., Rice, J. E., Sanchez, J. A., and Wangh, L. J. (2005) LATE-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection. Proc. Natl. Acad. Sci. USA, 102, 8609–8614.
Pierce, K. E., Rice, J. E., Sanchez, J. A., and Wangh, L. J. (2003) Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR. Mol. Hum. Reprod. 9, 815–820.
Pierce, K. E., Rice, J. E., Sanchez, J. A., and Wangh, L. J. (2002) QuantiLyse™: reliable DNA amplification from single cells. BioTechniques 32, 1106–1111.
Allawi, H. T. and SantaLucia, J. (1997) Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36, 10,581–10,584.
SantaLucia, J. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 5, 1460–1465.
Owczarzy, R., Vallone, P. M., Gallo, F. J., Paner, T. M., Lane, M. J., and Benight, A. S. (1998) Predicting sequence-dependent melting stability of short duplex DNA oligomers. Biopolymers 44, 217–223.
Breslauer, K. J. (1986) Methods for obtaining thermodynamic data on oligonucleotide transitions, in Thermodynamic Data for Biochemistry and Biotechnology (Hinz H. ed.), Springer-Verlag, New York, pp. 402–427.
Le Novère, N. (2001) MELTING, computing the melting temperature of nucleic acid duplex. Bioinformatics 17, 1226, 1227.
SantaLucia, J., Allawi, H. T., and Seneviratne, P. A. (1996) Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35, 3555–3562.
Wetmur, J. G. (1991) DNA probes: applications of the principles of nucleic acid hybridization. Crit. Rev. Biochem. Mol. Biol. 26, 227–235.
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.
Marras, S. A. E., Kramer, F. R., and Tyagi, S. (2003) Genotyping single nucleotide polymorphisms with molecular beacons, in Single Nucleotide Polymorphisms: Methods and Protocols, vol. 212 (Kwok, P. Y., ed.), Humana Press, Totowa, NJ, pp. 111–128.
Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160.
Pierce, K. E., Rice, J. E., Sanchez, J. A., Brenner, C., and Wangh, L. J. (2000) Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres. Mol. Hum. Reprod. 6, 1155–1164.
Rice, J. E., Sanchez, J. A., Pierce, K. E., and Wangh, L. J. (2002) Real-time PCR with molecular beacons provides a highly accurate assay for Tay-Sachs alleles in single cells. Prenat. Diagn. 22, 1130–1134.
Rosner, B. (1995) Fundamentals of Biostatistics, Wadsworth Publishing, Belmont, CA, pp. 277–282.
Rechitsky, S., Verlinsky, O., Amet, T., Rechitsky, M., Kouliev, T., Strom, C., and Verlinsky, Y. (2001) Reliability of preimplantation diagnosis for single gene disorders. Mol. Cell. Endocrinol. 183, S65–S68.
Al-Soud, W. A., Jonsson, L. J., and Radstrom, P. (2000) Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J. Clin. Microbiol. 38, 345–350.
Al-Soud, W. A. and Radstrom, P. (2001) Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 3, 485–493.
Cui, X. F., Li, H. H., Goradia, T. M., Lange, K., Kazazian, H. H. Jr, Galas, D., and Arnheim, N. (1989) Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers. Proc. Natl. Acad. Sci. USA 86, 9389–9393.
Li, Q., Luan, G., Guo, Q., and Liang, J. (2002) A new class of homogeneous nucleic acid probes based on specific displacement hybridization. Nucleic Acids Res. 30, e5.
Cheng, J., Zhang, Y., and Li, Q. (2004) Real-time PCR genotyping using displacing probes. Nucleic Acids Res. 32, e61.
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© 2007 Humana Press Inc., Totowa, NJ
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Pierce, K.E., Wangh, L.J. (2007). Linear-After-The-Exponential Polymerase Chain Reaction and Allied Technologies. In: Thornhill, A. (eds) Single Cell Diagnostics. Methods in Molecular Medicine™, vol 132. Humana Press. https://doi.org/10.1007/978-1-59745-298-4_7
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DOI: https://doi.org/10.1007/978-1-59745-298-4_7
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